Aflatoxin templates, molecularly imprinted polymers, and methods of making and using the same

ABSTRACT

Molecularly imprinted polymers (MIPs) are materials exhibiting molecular recognition of a target molecule. MIPs are synthesized in the presence of an aflatoxin template, a mimic to the targeted molecule, used as an imprint that is further washed away with suitable solvent after completion of the polymerization process, leaving a cavity in the polymer of the same stereochemistry, functionality and morphology to the template. When the MIP encounters an aflatoxin, the molecule is bound in the cavity with a receptor-like affinity.

FIELD OF THE DISCLOSURE

The disclosure relates generally to Aflatoxin templates and molecularlyimprinted polymers (MIPs). In particular, the disclosure relates toreusable, ecologically friendly MIPs, methods of producing the same, andmethods of utilizing the same (e.g., to sequester and/or adsorbaflatoxins). Compositions and methods of the disclosure find use in avariety of applications including dietary, therapeutic, prophylactic,food and beverage processing and manufacture, as well as research andquality control applications.

BACKGROUND

Mycotoxins are secondary metabolites secreted by a variety of fungi,often produced in cereal grains as well as forages before, during andafter harvest. Forages and cereals naturally come into contact withfungal spores. The fungal contamination of plants and the bio-synthesisof toxins depend on the state of health of the plant before harvest,meteorological conditions, harvesting techniques, delays andhydrothermal conditions before stabilization for conservation and feedprocessing. Depending on the fungus, fungal growth is controlled by anumber of physico-chemical parameters including the amount of free water(a_(w)), temperature, presence of oxygen, nature of the substrate, andpH conditions. Mycotoxins proliferate pre-harvest as well aspost-harvest in storage.

Some fungi produce toxins only at specific levels of moisture, wateravailability, temperature or oxygen. The effects of mycotoxins varygreatly in their severity. Some mycotoxins are lethal, some causeidentifiable diseases or health problems, some weaken the immune systemwithout producing symptoms specific to that mycotoxin, some act asallergens or irritants, and some have no known effect on animals orhumans. According to recent United Nation's Food and AgricultureOrganization (FAO) reports, approximately 25% of the world's grainsupply is contaminated with mycotoxins. Mycotoxin contamination has anegative economic impact on food and feed producers, particularly grainand animal producers.

Mycotoxins can appear in the food chain as a result of fungal infectionof plant products (e.g., forage, grain, plant protein, processed grainby-products, roughage and molasses products), and can either be eatendirectly by humans, or introduced by contaminated grains, livestock orother animal feedstuff(s). Mycotoxins greatly resist decompositionduring digestion so they remain in the food chain in edible products(e.g., meat, fish, eggs and dairy products) or under the form ofmetabolites of the parent toxin ingested. Temperature treatments such ascooking and freezing are not adequate methods of decreasing theprevalence of mycotoxins. Thus, there exists a need for compositionsand/or methods for reducing the detrimental effects and/or eliminatingmycotoxin occurrence in feed and/or food chains.

Aflatoxins are members of the mycotoxin family. These toxins areproduced by moulds of the Aspergillus sp. such as Aspergillus flavus orA. Parasiticus that contaminate a variety of feed and food materials andthat can ultimately transfer in their native form or has metabolites inanimal by-products such as milk, eggs or potentially meat. Aflatoxinsrepresent a significant health risk due to their high toxicity andcarcinogenicity and regulatory levels are strictly enforcing theiracceptable concentration in animal feeds and human food.

SUMMARY

There is a need for isolation of aflatoxins and metabolites frommaterials both for diagnostic and mitigation purposes. Molecularlyimprinted polymers (MIPs) as described herein are materials exhibitingmolecular recognition of an aflatoxin. MIPs are synthesized in thepresence of an aflatoxin template (e.g. a mimic of aflatoxin), which isused to make an imprint and then is removed from the polymer aftercompletion of the polymerization process, leaving a cavity in thepolymer of the same stereochemistry, functionality, and morphology ofthe template. When the MIP encounters the aflatoxin, the aflatoxin isbound in the cavity.

The present disclosure relates generally to aflatoxin templates andmolecularly imprinted polymers (MIPs). In particular, the disclosurerelates to reusable, ecologically friendly MIPs, methods of producingthe same, methods of utilizing the same (e.g., to sequester and/oradsorb aflatoxins), and methods for applying the use in different ways(e.g., to detect presence of aflatoxins for traceability purposes and toremove aflatoxins from a contaminated source). Compositions and methodsof the disclosure find use in a variety of applications includingdietary, therapeutic, prophylactic, food and beverage processing andmanufacture, liquid filtering as well as research and quality controlapplications.

In embodiments, aflatoxin templates, monomers, crosslinkers, and/or MIPshave favorable safety and/or environmental properties such as reduced orno toxicity, and high water sorption, and retention of aflatoxins. Inpreferred embodiments, MIPs can be reusable and economicallyrealizable/producible.

In one aspect of the disclosure, aflatoxin templates are provided. In aparticular embodiment, an aflatoxin template has a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl. In embodiments, R′ further comprises substituents selected from agroup consisting of halo, hydroxy and alkoxy. In a specific embodiment,an aflatoxin template is an isolated compound that has a Formula of:

Additional embodiments include aflatoxin templates that have a formulaselected from the group consisting of:

and combinations thereof.

Another aspect of the disclosure includes a method of synthesis of anaflatoxin template of Formula (I) comprising: reacting 3,5-dimethoxyphenol with ethyl 4-chloroacetoacetate in acid to form4-(2-chloroethyl)-5,7-dimethoxy coumarin.

In other embodiments, a method of synthesis of an aflatoxin templatecomprises suspending a monoacid according to the Formula of:

in polyphosphoric acid and heating to at least 50° C.; cooling thereaction mixture below 50° C. and adding an aqueous solution to obtainan aflatoxin template according to the Formula of:

In other embodiments, a monoacid is provided by suspending a diacidaccording to a Formula of:

in a solvent and heating to at least 100 to 140° C.

In another embodiment, a method of synthesis of an aflatoxin templatecomprises: deprotecting a diethyl intermediate2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate to form a diacidanalog; and precipitating the diacid analog to isolate the aflatoxintemplate according to the Formula of:

In other embodiments, a diethyl2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate is prepared by amethod comprising: combining 4-(2-chloroethyl)-5,7-dimethoxy coumarinwith diethyl malonate, potassium iodide, and a crown ether in a polarsolvent to form a mixture; and adding potassium butoxide to the mixtureto form diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate.In other embodiments, a method of synthesis of an aflatoxin template ofFormula (I) comprises: deprotecting diethyl2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate to form a diacidanalog, and precipitating the diacid analog according to the Formula:

Suspending the diacid analog in a solvent, heating to at least 100 to140° C., and precipitating the monoacid according to the Formula:

Suspending the monoacid in an acid and heating to at least 50° C.,cooling the reaction mixture to below 50° C., and adding an aqueoussolution to obtain a compound according to the Formula:

Another aspect of the disclosure provides a molecularly imprintedpolymer intermediate comprising a complex of a crosslinked polymer madefrom a monomer and an aflatoxin template having a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl, or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C₄₋₇ cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl. Inparticular embodiments, the molecularly imprinted polymer intermediatehas an aflatoxin template to monomer ratio from about 100:1 to 1:100. Inother embodiments, the molecularly imprinted polymer intermediate has amonomer to crosslinker ratio from about 1:4.1 to 1:10. In yet otherembodiments, the molecularly imprinted polymer intermediate includes anaflatoxin template of Formula (I) selected from the group consisting of4-(2-chloroethyl)-5,7-dimethoxy coumarin, 5,7-dimethoxycyclopentenon[2,3-c]coumarin, and combinations thereof.

Another aspect of the disclosure includes a molecularly imprintedpolymer comprising a crosslinked polymer made from a monomer, whereinthe polymer has a plurality of cavities, wherein at least one of thecavities was made using the aflatoxin template having a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl, or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C₄₋₇ cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl. Inparticular embodiments, the molecularly imprinted polymer includes anaflatoxin template that is 4-(2-chloroethyl)-5,7-dimethoxy coumarin. Inembodiments, the molecularly imprinted polymer includes an aflatoxintemplate that is 5,7-dimethoxycyclo pentenon[2,3-c]coumarin. Inembodiments, a molecularly imprinted polymer has a monomer that isselected from the group consisting of methacrylic acid, 2-vinylpyridine,2-hydroxyethylmethacrylate and combinations thereof. In embodiments, amolecularly imprinted polymer has a crosslinker that is ethylene glycoldimethacrylate. In yet other embodiments, the molecularly imprintedpolymer has aflatoxin template to monomer ratio that is from about 100:1to 1:100. In yet other embodiments, the molecularly imprinted polymerhas a monomer to crosslinker ratio that is from about 1:4.1 to 1:10.

Another aspect of the disclosure includes a method of making amolecularly imprinted polymer comprising the steps of: providing anaflatoxin template having a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl, or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C₄₋₇ cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl;combining the aflatoxin template with at least one monomer and one ormore crosslinkers; polymerizing the monomer and the one or morecrosslinkers to form a molecularly imprinted polymer intermediate; andremoving the aflatoxin template from the molecularly imprinted polymerintermediate to form a molecularly imprinted polymer. In particularembodiments, the aflatoxin template comprises5,7-dimethoxy-cyclopentenon [2,3-c]coumarin4-(2-chloroethyl)-5,7-dimethoxy coumarin, or combinations thereof. Inembodiments, an MIP is prepared by a process as described herein.

In embodiments, the step of combining of aflatoxin template compoundwith at least one monomer and one or more crosslinkers comprises mixingthe monomer and the crosslinker in a solution of one or more organicsolvents. In particular embodiments, the one or more solvents areselected from the group consisting of acetonitrile, toluene,cyclohexane, polyvinyl alcohol in water solution, and a mixture of twoor more of acetonitrile, toluene, cyclohexane, polyvinyl alcohol inwater solution.

In other embodiments, a method further comprises adding an initiator. Ina particular embodiment, the initiator is azo(bis)-isobutyronitrile(AIBN), wherein free radicals are formed by thermal decomposition ofAIBN acting as an initiator. In yet other embodiments, polymerization isinitiated by forming free radicals in an organic solvent at atemperature between 55 and 110° C.

In embodiments, the removal of the aflatoxin template from themolecularly imprinted polymer intermediate comprises washing themolecularly imprinted polymer intermediate with a solvent. In particularembodiments, the organic solvent is selected from the group of ethylalcohol, methyl alcohol, acetonitrile, toluene, and a mixture ofthereof.

In embodiments, the molecularly imprinted polymer is dried after saidone or more washes.

Another aspect of the disclosure includes a method of sequestering anaflatoxin comprising: providing a molecularly imprinted polymer having aplurality of cavities, wherein at least one of the cavities is madeusing the aflatoxin template having a Formula I:

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl, or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C₄₋₇ cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl;providing a material, wherein the material optionally contains anaflatoxin; and contacting the molecularly imprinted polymer with thematerial.

In embodiments, the material is a liquid, a solid, or a gas. Inparticular embodiments, the material is selected from the groupconsisting of soil, a spice, a beverage, a foodstuff, an animal feed, apharmaceutical composition, a nutraceutical composition, and a cosmeticcomposition. In a specific embodiment, the material is milk.

In embodiments, the molecularly imprinted polymer is contacted with thematerial for at least 1 second.

In embodiments, a method further comprises separating the molecularlyimprinted material from the material. In particular embodiments, theseparation of the molecularly imprinted polymer comprises separating byfiltration or by centrifugation.

In embodiments, a method further comprises detecting an amount ofaflatoxin complexed with the molecularly imprinted polymer, detectingthe amount of aflatoxin in the material after contact with themolecularly imprinted polymer, or both.

In other embodiments, a method of sequestering an aflatoxin comprisessteps of: providing a molecularly imprinted polymer having a pluralityof cavities, wherein at least one cavity was made using an aflatoxintemplate having a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl, or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C₄₋₇ cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl; b)providing a material containing an aflatoxin; and c) contacting themolecularly imprinted polymer with the material, wherein the molecularlyimprinted polymer sequesters at least 40 percent of the aflatoxin byweight per unit of the material. In embodiments, the material is aliquid and the molecularly imprinted polymer sequesters at least 40percent of the weight of aflatoxin per volume of the material.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the quantity of aflatoxin M1 (AFM1) adsorbed by 1 mgquantity of each MIP in an instant trapping solid phase extraction (SPE)column setup using 1 mL of a 100 ng/L solution of AFM1 in a pH 6.0ammonium acetate buffer (

). Also shown are the quantities of AFM1 present in subsequent methanol(

) and toluene (▪) washed performed after the adsorption step. Quantityof AFM1 adsorbed vs. quantity of AFM1 released from the material can bequantified.

FIG. 2 shows average AFM1 adsorption averaged over multiple time pointsfor the indicated MIPs and corresponding non-imprinted polymers(NIPs)(varying from 0.001%-0.1%)sing free flowing MIP/NIP over 6 periodsof time, from 5 to 500 minutes with a 90 ng/L AFM1 10 mL solution.Adsorption efficacy was measured by quantitation of the mycotoxinremaining in the supernatant (

) and eluting from the MIP/NIP after methanol wash (

), which defined adsorption efficacy and selectivity. Due to the factthat there is instant adsorption, the AFM1 adsorption quantities foreach time point (15, 30, 60, 90 minutes and 18 hrs) were averaged foreach product. All test tubes were then centrifuged for 10 minutes at3,000 rpm and a transferred into UPLC vial for analysis. The powder (MIPor NIP) was then transferred to a 2 mL Eppendorf tube where 1 mL ofmethanol was added and vortexed for approximately three seconds.

FIG. 3 shows average AFM1 adsorption averaged for MIP-003 (varying from0.001%-0.1%) in 10 mL of raw milk spiked with a concentration of 225ng/L AFM1.

FIG. 4 is a schematic diagram of a synthesis of an aflatoxin template.

FIG. 5 shows results for the instant trapping of AFM1 by MIP-005 atvarious inclusion rates (ranging from 0.001%-0.1%) in an SPE columnsetup at room temperature.

DEFINITIONS

As used herein, the term “about,” when used in reference to a particularrecited numerical value, means that the value may vary from the recitedvalue by no more than 1%. For example, as used herein, the expression“about 100” includes 99 and 101 and all values in between (e.g., 99.1,99.2, 99.3, 99.4, etc.).

As used herein, the term “molecularly imprinted polymer(s)” or “MIP(s)”refers to synthetic polymers that selectively bind to one or moreaflatoxins. In embodiments, a MIP exhibits high enantioselectivity andlow substrate selectivity, wherein the MIP interacts with the templateaflatoxin racemate as well as its corresponding analogs, such asnaturally occurring aflatoxins. In embodiments, the MIP has a higherenantioselectivity than the corresponding non-imprinted polymer for thebinding of aflatoxins. In embodiments, the MIP selectively binds toaflatoxins and does not bind to other mycotoxins. In embodiments, theMIP binds to one or more of aflatoxins B1, B2, G1, G2, M1, M2, P1, andQ1. In embodiments, the MIP selectively binds aflatoxin B1 and aflatoxinM1.

In embodiments, a polymer is crosslinked to generate cavities, at leastone of which is made using an aflatoxin template of Formula (I). In someembodiments, at least one cavity provides a site of interaction forreversible binding with an aflatoxin template of Formula (I) and/oraflatoxins. In general, MIPs are constructed using: i) a templates(e.g., aflatoxin template) that mimic the structure, size, shape and/orother chemical characteristics of one or more targeted compound(s)(e.g., aflatoxins) and ii) other components, such as monomers and/orcross linking reagents. For example, one or more aflatoxin templates areincorporated into a pre-polymeric mixture comprising monomers and acrosslinker. The mixture is then polymerized to form a “molecularlyimprinted polymer intermediate” or “MIP intermediate” comprising acrosslinked polymer and the aflatoxin template(s). Once the polymer hasformed, the aflatoxin template(s) is/are removed, leaving behindcomplementary cavities having a chemical and/or physical capacity toform a complex with one or more aflatoxins or other compounds resemblingaflatoxin. Such regions (e.g., cavities or other regions) are tailoredfor binding one or more aflatoxins giving rise to a high affinity forsuch aflatoxin containing compounds and selectivity. While aflatoxintemplate compounds are used to form molecularly imprinted polymers, insome embodiments, the MIPs may have a high affinity for a class ofcompounds that is distinct from but similar to one or more aflatoxins.For example, a MIP may bind a number of compounds containing moleculesthat are similar in shape, size, charge density, geometry or otherphysical or chemical properties to one or more aflatoxins.

As used herein, the term “non-imprinted polymer” or “NIP” refers tosynthetic polymers that are formed without the presence of a templatecompound (e.g., aflatoxin template). Such polymers, which have noenantioselectivity nor substrate selectivity, and might interact withany molecules susceptible to generate hydrogen-bounding, ionicinteraction, electrostatic interaction with the components of the NIP.NIPs are involved in non-specific non-covalent surface interactions oflower stability than when a specific cavity is available from theimprinting process in the MIP network to target a specific compound(e.g., AFB1, AFM1). In embodiments, a “corresponding” NIP refers to asynthetic polymer synthesized with the same monomer and crosslinker as aMIP but without the use of a template (e.g., aflatoxin template).

As used herein, the term “polymer”, refers to a molecule (macromolecule)composed of repeating structural units (e.g. monomer) typicallyconnected by covalent chemical bonds forming a network. In embodiments,a polymer is formed by crosslinking of monomers forming primary chainsor structural units that assemble in a network.

As used herein, the term “aflatoxin template(s)” refer(s) to one or moresynthetically constructed molecule(s) that mimic the structure, size,shape and/or other chemical characteristics of one or more naturalaflatoxins. The disclosure is not limited by the type of aflatoxintemplate utilized, that can be either synthetic or natural. Indeed avariety of aflatoxins may bind to MIPs generated using an aflatoxintemplate compound including, but not limited to, aflatoxin B1, B2, G1,G2, M1, P1, Q1, and other aflatoxins described herein.

As used herein, the term “monomer(s)”, refers to a molecule that maybecome chemically bonded to other monomers to form a polymer.

As used herein, the terms “crosslink” and “crosslinker”, refer tomolecules that contain two, three or four double-bonds that are capableof attaching to two or more monomers to form a polymer network.

As used herein, the term “structural unit”, refers to a building blockof a polymer chain, and related to the repeat unit.

As used herein, the term “anionic” or “anion” refers to an ion that hasa negative charge.

As used herein, the term “cationic” or “cation” refers to an ion thathas a positive charge. This term can refer to polymeric compounds, suchas molecularly imprinted polymers, that contain a positive charge.

As used herein, the term “acid” as used herein refers to any chemicalcompound that can donate proton(s) and/or accept electron(s). As usedherein, the term “base” refers to any chemical compound that can acceptproton(s) and/or donate electron(s) or hydroxide ions. As used herein,the term “salt” refers to compounds that may be derived from inorganicor organic acids and bases.

As used herein, the term “bleeding”, refers to a remaining fraction ofthe template still in association with the MIP after several washingstages of the MIP, and that continues to dissociate from the MIP andinterfere with its adsorption activity.

As used herein, the term “porogenic/porogen”, refers to a substance,molecule, buffer, solvent, (e.g., toluene, xylene, ethylbenzene) used tochange the size of the cavities in a polymer (e.g., cavities of a MIP).In embodiments, a polymer to porogen ratio is directly correlated to theamount of porosity of the final structure and dictates the size of thepolymer agglomerates formed.

As used herein, the term “inclusion rate” refers to the amount of MIPprovided per unit of material (e.g. milk), for example, in a unit ofweight of the polymer as compared to a unit of volume of the material orin a unit of weight of the polymer as compared to a unit per weight ofthe material.

As used herein, the term “cavity(ies)”, refer(s) to a space, pore, orother opening that is/are within the MIP and that are sized and/orshaped to allow an aflatoxin to be bound therein. In embodiments, acavity is formed in a crosslinked polymer by polymerizing the polymer inthe presence of the aflatoxin template and removing the aflatoxintemplate to form the cavity in the crosslinked polymer, which is now anMIP.

As used herein, the term “polymerization”, refers to a process ofreacting monomer molecules together in a chemical reaction to formthree-dimensional networks or polymer chains and agglomerated polymerschains.

As used herein, the term “precipitation”, refers to the formation of asolid in a solution during a chemical reaction. When the reactionoccurs, the solid formed is called the precipitate, and the liquidremaining above the solid is called the supernatant.

As used herein, the term “centrifugation” refers to the process ofseparating molecules by size or density using centrifugal forcesgenerated by a spinning rotor that puts an object in rotation around afixed axis, applying a force perpendicular to the axis. The centrifugeworks using the sedimentation principle, where the centripetalacceleration is used to evenly distribute substances of greater andlesser density into different layers of density.

As used herein, the term “concentration” refers to the amount of asubstance per defined space. Concentration usually is expressed in termsof mass per unit of volume. To dilute a solution, one must add moresolvent, or reduce the amount of solute (e.g., by selective separation,evaporation, spray drying, freeze drying). By contrast, to concentrate asolution, one must reduce the amount of solvent.

As used herein, the term “layer” refers to a usually horizontal depositorganized in stratum of a material forming an overlying part or segmentobtained after separation by centrifugation or sedimentation in relationwith the density properties of the material.

As used herein, the term “purified” or “to purify” refers to the removalof foreign components from a sample. When used in a chemical context“purified” or “to purify” refers to the physical separation of achemical substance of interest from undesired substances. Commonly usedmethods for purification of organic molecules, include, but are notlimited to the following: affinity purification, mechanical filtration,centrifugation, evaporation, extraction of impurity, dissolving in asolvent in which other components are insoluble, crystallization,adsorption, distillation, fractionation, sublimation, smelting,refining, electrolysis and dialysis.

As used herein, the term “drying” refers to any kind of process thatreduces or eliminates the amount of liquid in a substance.

As used herein, the term “washing” refers to the removal (e.g., usingany type of solute (e.g., distilled water, buffer, or solvent, ormixture)) of impurities or soluble unwanted component of a preparation(e.g., a MIP may be washed to remove the aflatoxin template componentsfrom the sample).

As used herein, the term “analyte” refers to an atom, a molecule, asubstance, or a chemical constituent. In general, an analyte, in and ofitself is not measured, rather, aspects or properties (physical,chemical, biological, etc.) of the analyte are determined using ananalytical procedure, such as Ultra Performance Liquid Chromatography(abbreviated as UPLC). For example, in general one does not measure a“chair” (analyte-component) in and of itself, but, the height, width,etc. of a chair are measured. Likewise, in general one does not measurean aflatoxin but rather measures one or more properties of the aflatoxin(e.g., aflatoxins fluorescence or molecular weight, related for example,to its stability, concentration, or biological activity).

As used herein, the term “sample” is used in a broad sense including aspecimen from any source (e.g., synthetic, biological and environmentalsamples). Synthetic samples include any material that is artificiallyproduced (e.g., MIP). Biological samples may be obtained from animals(including humans) and encompass fluids, solids, tissues, and gases.Biological samples include blood products, such as plasma, serum and thelike. Environmental samples include environmental material such assurface matter, soil, water, crystals and industrial samples.

As used herein, the term “Ultra Performance Liquid Chromatography” or“UPLC” refers to a form of liquid chromatography to separate compounds.The compounds are dissolved in a solution. Compounds are separated byinjecting a sample mixture onto a column, through which a solvent orsolvent mixture has been flowing at a specific pressure, to elutecomponents of the mixture, from the column. UPLC instruments compriseone or more reservoirs of mobile phases, a pump, an injector, aseparation column, and a detector. The presence of analytes in thecolumn effluent is recorded by quantitatively detecting a change inrefractive index, UV-VIS absorption at a set wavelength, fluorescenceafter excitation with a suitable wavelength, electrochemical response,or mass to charge ratio based on the molecular weight of an analyte in acharged state.

As used herein, the term “signal” is used generally in reference to anydetectable process that indicates that a reaction has occurred (forexample, binding of antibody to antigen). Signals can be assessedqualitatively as well as quantitatively. Examples of types of“signals”include, but are not limited to, radioactive signals, fluorometricsignals, colorimetric product/reagent signals, mass to charge ratiomeasure.

As used herein, the terms “absorb” and “absorption” refer to the processby which a material “takes in” or “sucks up” another substance. Forexample, “absorption” may refer to the process of taking in orassimilating substances into cells or across the tissues and organsthrough diffusion or osmosis (e.g., absorption of nutrients by thedigestive system or absorption of drugs into the blood stream).

As used herein, the terms “adsorb” and “adsorption” refer to a processthat occurs when a material is captured by, sequestered by, bound by,trapped by, and/or accumulated by (e.g., on the surface of) acomposition (adsorbent), or to a process in which a composition (e.g.,MIP) binds to a target molecule (e.g., one or more aflatoxins) in asample (e.g., for removing the target molecule from a sample).

As used herein, the terms “sorb” and “sorption” refer to both adsorptionand absorption.

As used herein, the terms “sequester”, “capture”, “trap”, “adsorb”, or“bind” refer to physical association (e.g., via bonding (e.g., hydrogenboding, ionic bonding, covalent bonding or other type of bonding) of twoor more entities that come into contact with one another (e.g., therebyforming a complex). Exemplary forms of associations include, but are notlimited to, hydrogen bonding, coordination, and ion pair formation.Sequestering interactions may involve a variable number of chemicalinteractions (e.g., chemical bonds) depending on the stereochemistry andgeometry of each entity (e.g., further defining the specificity of thesequestering). When two or more entities are interacting they may besequestered by way of chemical bonds or physical bonds but may also beassociated via charge, dipole-dipole or other type of interactions.

As used herein, the terms “sequestering agent”, “capturing agent”,“trapping agent”, “adsorbing agent” and/or “binding agent”, refer to anentity that is capable of forming a complex with a second entity.

As used herein, the term “complex” refers to an entity formed byassociation between two or more separate entities (e.g., associationbetween two or more entities wherein the entities are the same ordifferent (e.g., same or different chemical species).

The association may be via a covalent bond or a non-covalent bond (e.g.,via van der Waals, electrostatic, charge interaction, hydrophobicinteraction, dipole interaction, and/or hydrogen bonding forces (e.g.,urethane linkages, amide linkages, ester linkages, and combinationthereof)).

As used herein, the term “bind” refers to a close association betweentwo or more separate entities (e.g., association between two or moreentities wherein the entities are the same or different (e.g., same ordifferent chemical species). The association may be via a covalent bondor a non-covalent bond (e.g., via van der Waals, electrostatic, chargeinteraction, hydrophobic interaction, dipole interaction, and/orhydrogen bonding forces (e.g., urethane linkages, amide linkages, esterlinkages, and combination thereof)). As used herein, the term “close”refers to touching or near touching.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., MIP) sufficient to accomplish beneficial or desiredresults. An effective amount can be administered and/or combined withanother material in one or more administrations, applications or dosagesand is not intended to be limited to a particular formulation oradministration route.

As used herein, the term “animal” refers to any one or more species inthe kingdom of animalia. This includes, but is not limited to livestock,other farm animals, domestic animals, pet animals, marine and freshwateranimals, and wild animals.

As used herein, the term “feedstuffs” refers to material(s) that areconsumed by a human or animal that contribute energy and/or nutrients tothe subject. Examples of feedstuffs include, but are not limited to,dairy products, juices, grains, including but not limited to distillersgrains, fruits, vegetables, meats, Total Mixed Ration (TMR), forage(s),pellet(s), concentrate(s) of any of the previous items, premix(es) orcoproduct(s) of any of the previous products, molasses, fiber(s),fodder(s), grass(es), hay, kernel(s), leaves, meals made from any of theprevious products, soluble(s) and supplement(s) containing any of theprevious products.

As used herein, the term “mycotoxin” refers to toxic and/or carcinogeniccompound(s) produced by various fungal species. In embodiments, themycotoxin is an aflatoxin.

As used herein, the term “mycotoxicosis” refers to a condition in whichmycotoxins pass the resistance barriers of a human or animal body.Mycotoxicosis can be considered either an infection or a disease and mayhave a deleterious effect on those afflicted.

As used herein, the term “toxic” refers to any detrimental, deleterious,harmful, or otherwise negative effect(s) on an animal or human,including, but not limited to a cell or a tissue of such animal orhuman. As used herein the terms “detrimental”, “deleterious”, “harmful”,or “otherwise negative” with respect to “effect” can be determined bycomparing the same cell or tissue of an animal or human prior to thecontact or administration of a toxin or toxicant and after such contactand detecting an undesirable change in such cell or tissue when makingsuch comparison.

As used herein, the term “traceability” refers to the property of theresult of a measurement or the value of a standard whereby it can berelated to stated references, usually national or internationalstandards, through an unbroken chain of comparisons, all having stateduncertainties. It is the practical application of general metrologyconcepts to chemical measurements and provides the terminology, conceptsand strategy for ensuring also that analytical chemical measurements arecomparable. It measures the uniquely identifiable entities in a way thatis verifiable. Traceability measures are utilized, among other things,to interrelate the chronology, location, and/or application of an itemby means of documented recorded identification.

As used herein, the term “alkyl”, by itself or as part of anothersubstituent, refers to, unless otherwise stated, a straight or branchedchain, or cyclic hydrocarbon radical, or combination thereof, which isfully saturated, having the number of carbon atoms designated (e.g.,C1-C6 means one to six carbons). Examples of alkyl groups include, butare not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, cyclohexyl, homologs and isomers of, for example,n-pentyl, n-hexyl, and the like.

As used herein, the term “heteroalkyl”, by itself or as part of anothersubstituent, refers to, unless otherwise stated, a straight or branchedchain, or cyclic hydrocarbon radical, or combination thereof, which isfully saturated, having the number of carbon atoms designated (e.g.,C1-C6 means one to six carbons) in which one of the carbon atom isreplaced by a heteroatom. In embodiments a heteroatom is an oxygen.

As used herein, the term “substituted alkyl”, unless otherwise stated,refers to a straight or branched chain, or cyclic hydrocarbon radical,or combination thereof, which is fully saturated, having the number ofcarbon atoms designated (e.g., C1-C6 means one to six carbons) andhaving a substitution of at least one of the H atoms. Examples of alkylgroups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, cyclohexyl, homologs and isomers of, for example, n-pentyl,n-hexyl, and the like.

Examples of substituents that can be used in a substituted alkylinclude, but are not limited to, halogens, carboxy, and hydroxyl groups.As used herein, the term “halo substituted alkyl”, by themselves or incombination with other terms, unless otherwise stated, refers to asubstituted alkyl wherein a halo atom is used to replace at least one ofthe H atoms.

As used herein, the terms “cycloalkyl” and “heterocycloalkyl” bythemselves or in combination with other terms, refer to, unlessotherwise stated, cyclic versions of “alkyl” and “heteroalkyl”respectively. Additionally, for heterocycloalkyl, a heteroatom canoccupy the position at which the heterocycle is attached to theremainder of the molecule.

The terms “halo” or “halogen,” by themselves or in combination withother terms, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

Additionally, terms such as “haloalkyl”, are meant to include one ormore substituted alkyl groups with halogen atoms that can be the same ordifferent, in a number ranging from one to (2m+1), where m is the totalnumber of carbon atoms in the alkyl group.

Thus, the term “haloalkyl” includes monohaloalkyl (alkyl substitutedwith one halogen atom) and polyhaloalkyl (alkyl substituted with halogenatoms in a number ranging from two to (2m+1) halogen atoms).

The term “alkoxy,” refers to one or more alkyl groups attached to theremainder of the molecule via an oxygen atom.

DETAILED DESCRIPTION

This disclosure describes aflatoxin template(s), compounds containingone or more such aflatoxin templates, and molecularly imprinted polymersmade using such compounds, and methods of making and using suchtemplates and compounds.

Aflatoxin Template(s) and Intermediates

In embodiments, aflatoxin templates described herein are structuralanalogs to aflatoxin molecules. In other embodiments, aflatoxintemplate(s) are similar in shape, size, charge density, geometry and/orother physical or chemical properties to one or more aflatoxins. Inspecific embodiments, an aflatoxin template comprises a coumarin moiety,at least one alkoxy moiety, and a carbonyl moiety. Aflatoxins moleculesinclude one or more types of aflatoxin B1, B2, G1, G2, M1, M2, P1, andQ1.

In embodiments, Aflatoxin B1 (AFB1) was used as a model to create astructural analog using the synthesis described herein. Aflatoxinanalogs are advantageous because they reduce or eliminate the need tohandle large quantities of toxic aflatoxins and to prevent bleeding ofaflatoxins out of the polymer. In embodiments, at least two differentaflatoxin templates are prepared. In embodiments, aflatoxin templateshave reduced or no toxicity as compared to naturally occurringaflatoxins.

An aflatoxin template has or comprises a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂, R′ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,and a halo substituted C₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆alkoxy, and substituted C₁₋₆ alkyl. In related embodiments, R′ is one ormore substituents selected from a group consisting of halo, oxo, hydroxyand alkoxy.

In embodiments, R₁, R₂, R₃, and R′ may independently be alkyl groups. Analkyl, refers to a straight or branched chain, or cyclic hydrocarbonradical, or combination thereof, which is fully saturated, having thenumber of carbon atoms designated (e.g., C1-C6 means one to sixcarbons). Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, cyclohexyl, homologs and isomersof, for example, n-pentyl, n-hexyl, and the like.

In embodiments, R₁, R₂, R₃, and R′ may independently be substitutedalkyl groups. A substituted alkyl is a straight or branched chain, orcyclic hydrocarbon radical, or combination thereof, which is fullysaturated, having the number of carbon atoms designated (e.g., C1-C6means one to six carbons) and having a substitution of at least one ofthe H atoms. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, cyclohexyl, homologs and isomersof, for example, n-pentyl, n-hexyl, and the like. Examples ofsubstitutions include halogens, carboxy, and hydroxyl groups.

In embodiments, R₁, R₂, R₃, and R′ may independently be a halogen. Ahalo group or halogen, refers to a fluorine, chlorine, bromine, oriodine atom. Additionally, haloalkyl includes alkyl substituted with oneor more halogen atoms, each of which can be the same or different, in anumber ranging from one to (2m+1), where m is the total number of carbonatoms in the alkyl group. Examples include monohaloalkyl (alkylsubstituted with one halogen atom) and polyhaloalkyl (alkyl substitutedwith halogen atoms in a number ranging from two to (2m+1) halogenatoms). In embodiments, the halogen is a chlorine or fluorine.

In embodiments, R₃ may independently be an alkoxy. An alkoxy refers tothose alkyl groups attached to the remainder of the molecule via anoxygen atom. Examples include methoxy, ethoxy and the like.

In some example embodiments, an aflatoxin template has or comprises theFormula:

In a specific embodiment, an aflatoxin template is4-(2-chloroethyl)-5,7-dimethoxy coumarin.

In other related embodiments, an aflatoxin template comprises anisolated compound having a formula selected from the group consisting of

and combinations thereof.

In embodiments, an aflatoxin template comprises an isolated compoundselected from the group consisting of2-((5,7-dimethoxy-2-oxo-2H-chromen-4yl)methyl) malonic acid,3-(5,7-dimethoxy-2-oxo-2H-chromen-4yl)propanoic acid, diethyl2-((5,7-dimethoxy-2-oxo-2H-chromen-4yl)methyl) malonate and combinationsthereof.

In an alternative embodiment, an aflatoxin template has or comprisesFormula

wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, a halosubstituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇ cycloalkylring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇ cycloalkyl ringand a carboxylic group substituted C₄₋₇ cycloalkyl; and R₃ is selectedfrom H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl.

In embodiments, cycloalkyl and heterocycloalkyl represent, cyclicversions of alkyl and heteroalkyl respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule.

In other related embodiments, an aflatoxin template has or comprises theFormula:

In a specific embodiment, the aflatoxin template is 5,7-dimethoxycyclopentenon[2,3-c]coumarin.

Aflatoxin Template Synthesis

Aflatoxin templates and compounds containing such aflatoxin templates asdescribed herein can be prepared by a variety of methods. The exemplarymethods described herein provide processes (e.g., a synthetic process)and materials that allow large scale production of compounds containingone or more aflatoxin templates that are not only economical (e.g., thatenables realizable, large scale production in an economically achievablemanner), but that also use reagents that generally can be more readilyavailable than reagents used to make mycotoxin templates previously.

In embodiments, a method of synthesis of an aflatoxin template ofFormula (I) comprises reacting 3,5-dimethoxy phenol with ethyl4-chloroacetoacetate in acid to form 4-(2-chloroethyl)-5,7-dimethoxycoumarin. In other embodiments, the compound4-(2-chloroethyl)-5,7-dimethoxy coumarin is isolated and is used to forma MIP.

In embodiments, a method of synthesis of an aflatoxin template comprisessuspending a monoacid according to the Formula of:

in polyphosphoric acid and heating to at least 50° C.; cooling thereaction mixture below 50° C. and adding an aqueous to obtain anaflatoxin template according to the Formula of:

This aflatoxin template is isolated and used to form a MIP.

In embodiments, a method of providing a monoacid comprises suspending adiacid according to a Formula of:

in a solvent and heating to at least 100° C., or about 100 to 140° C.

In embodiments, a method of synthesis of an aflatoxin template comprisesdeprotecting a diester analog (i.e.2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate) according to aFormula of:

using a base (e.g. NaOH) in a solvent (e.g. ethanol) and heating to atleast 60° C.; to form a diacid analog; and precipitating the diacidanalog to isolate the aflatoxin template according to a Formula of:

In embodiments, a method of synthesis of diester intermediate (i.e.,diethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate)comprises combining 4-(2-chloroethyl)-5,7-dimethoxy coumarin accordingto a Formula of:

with diethyl malonate, potassium iodide, and a crown ether to form adiester analog; and precipitating the diester analog to isolate theaflatoxin template intermediate according to a Formula of:

In embodiments, a method of synthesis of an aflatoxin template ofFormula (I) comprises deprotecting diethyl2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate to form a diacidanalog, and precipitating a diacid analog according to a Formula of:

suspending the diacid analog in a solvent and heating to 100° C., or 100to 140° C. and precipitating a monoacid according to a Formula of:

suspending the monoacid in an acid and heating to at least 50° C.,cooling the reaction mixture to below 50° C., and adding aqueoussolution to obtain

Referring now to FIG. 4 where aflatoxin template analogs andintermediate compounds were formed by condensation of3,5-dimethoxyphenol with ethyl-4-chloroacetoacetate in presence of H₂SO₄in toluene to form a chlorinated analog, 4-(2-chloroethyl)-5,7-dimethoxycoumarin. In this example, 4-(2-chloroethyl)-5,7-dimethoxy coumarin iscombined with diethyl malonate, potassium iodide, and a crown ether inacetonitrile to form a mixture. Once the mixture is formed, potassiumt-butoxide is added to the mixture to form a diester with the Formuladiethyl 2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate.

Upon the formation of the diethyl2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate, in thisembodiment, a diacid is formed by deprotecting diethyl2-((5,7-dimethoxy-2-oxo-2H-chromen-4-yl)methyl)malonate using a base inalcohol. The diacid, having a Formula of:

is heated to at least 135° C. in a solvent. In this embodiment, thediacid is then converted to its monoacid by partial decarboxylation inxylene at reflux temperature. In at least this embodiment, the monoacidwas subjected to cyclization using polyphosphoric acid to yield thefinal AFT-1 aflatoxin template (AFT-1) with the Formula of:

It should be appreciated that the chemical formula used, must allow formolecularly imprinted polymer intermediates, described in further detailbelow, to reversibly bind the aflatoxin template to the MIP.Additionally, the aflatoxin template contained in the aflatoxin templatemust provide a molecularly imprinted polymer intermediate with a cavitythat retains a high level of affinity for one or more aflatoxins, suchas aflatoxin B1.

In embodiments, a composition comprising an aflatoxin template and acarrier is provided. In embodiments, the composition includes aneffective amount of the aflatoxin template to form a MIP with thedesired characteristics (e.g. typically represented as an amount inrelation to the amount of the monomer, a ratio). The compositions areformulated with suitable carriers, excipients, and other agents thatprovide suitable transfer, delivery, stability, and functionality of theaflatoxin template.

Molecularly Imprinted Polymers

In embodiments, a molecularly imprinted polymer comprises a crosslinkedpolymer comprising a monomer or made from a monomer, wherein thecrosslinked polymer has a plurality of cavities, and at least one of thecavities is made with an aflatoxin template having a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl; or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C4-7 cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl.

In embodiments, at least one cavity provides for binding of theaflatoxin template of Formula (I). In an embodiment, a MIP selectivelybinds one or more aflatoxin templates. In embodiments, a MIP selectivelybinds one or more types of aflatoxins, for example, aflatoxins B1, B2,G1, G2, M1, M2, P1, and Q1. In embodiments, the affinity and/orselectivity of the MIP for an aflatoxin is compared to a correspondingNIP.

In some cases, all or a portion of the binding of the aflatoxin templateor aflatoxin is reversible under certain conditions. After an MIPintermediate is formed, an aflatoxin template is removed using asolvent. In embodiments, a solvent is selected that can disrupt theinteraction of the aflatoxin template with the polymer and has a similarpolarity and/or solubility as the aflatoxin template. In embodiments,the solvent is a polar solvent.

Alternatively, after the MIP has bound aflatoxin from a material and isseparated from the material, in embodiments, the bound aflatoxin can beremoved in order to reuse the MIP. In embodiments, at least a portion ofthe bound aflatoxin and/or aflatoxin template is removable from the MIPusing a solvent, such as a polar solvent. In embodiments, a solvent isselected that can disrupt the interaction of the aflatoxin with thepolymer and has a similar polarity and/or solubility as the aflatoxin.In embodiments, a solvent is selected from the group of ethyl alcohol,methyl alcohol, acetonitrile, toluene, and a mixture of thereof. In someembodiments, about 25% or less of the aflatoxin bound to the MIP isreleased based on weight per volume in the presence of a solvent. Inembodiments, the MIP releases 25%, 20%, 15%, 10%, 5%, or 1% or less byweight of one or more aflatoxins sequestered from a material, forexample, in polar solvent. In contrast, about 90% or more aflatoxin isreleased from a corresponding NIP in the presence of the same solvent.

In embodiments, the MIP binds to the aflatoxin template with a chemicaland/or physical interaction. In other embodiments, the polymer networkforming the cavities binds to the aflatoxin template with a covalent ornoncovalent bond. In embodiments, a MIP comprises micropores of about 20Angstroms or less, and/or meso- and macropores between about 20 and 2000Angstroms. In embodiments, an aflatoxin template has a molar volume ofat least 300 cubic Angstroms. In other embodiments, the MIP has one ormore cavities or pores that have a molar volume of at least 300 cubicAngstroms.

In embodiments, an aflatoxin template has a formula of Formula (I) asdescribed herein. In a specific embodiment, the aflatoxin template ofFormula (I) is selected from the group consisting of4-(2-chloroethyl)-5,7-dimethoxy coumarin, 5,7-dimethoxycyclopentenon[2,3-c]coumarin, and combinations thereof. In embodiments, amolecularly imprinted polymer is synthesized using more than one of theaflatoxin templates of Formula (I).

In embodiments, a polymer is formed from a monomer. A monomer isselected taking into account structural features of the aflatoxintemplate in order to assess which monomer or combination of monomers ismost likely to form interactions (e.g., covalent, non-covalent, ionic,hydrogen bonds, hydrophobic interactions, van der Waals interactions)with the template. In the case of polymeric or oligomeric compounds thatare to be utilized in vivo (e.g., as therapeutics or diagnostics, or asconsumable sequestering components of animal feed or human foodstuffs),it is important to select monomers that are non-toxic and which exhibitsuitable in vivo stability and solubility. Preferred examples for anaflatoxin MIP include, but are not limited to, acrylamides andmethacrylates. Alternatively, the polymer may be treatedpost-polymerization to enhance the template solubility, e.g., byreaction with suitable organic or inorganic reagents.

Classes of monomers and specific monomers (e.g., utilized in MIPsynthesis methods of the disclosure) include, but are not limited to,the following classes and derivatives thereof: acrylic acid andderivatives (e.g., 2-bromoacrylic acid, acryloyl chloride, N-acryloyltyrosine, N-acryoyl pyrrolidinone, trans-2-(3-pyridyl)-acrylic acid),acrylates (e.g., alkyl acrylates, allyl acrylates, hydroxypropylacrylate), methacrylic acid and derivatives (e.g., itaconic acid,2-(trifluoromethyl) propenoic acid), methacrylates (e.g., methylmethacrylate, hydroxyethyl methacrylate, 2-hydroxyethyl methacrylate,3-sulfopropyl methacrylate sodium salt, ethylene glycolmonomethacrylate), styrenes (e.g., (2, 3 and 4)-aminostyrene,styrene-4-sulfonic acid, 3-nitrostyrene, 4-ethystyrene), vinyls (e.g.,vinyl chloroformate, 4-vinylbenzoic acid, 4-vinylbenzaldehyde, vinylimidazole, 4-vinylphenol, 4-vinylamine, acrolein), vinylpyridines (e.g.,(2, 3, and/or 4)-vinylpyridine, 3-butene 1,2-diol), boronic acids (e.g.,4-vinylboronic acid), sulfonic acids (e.g., 4-vinylsulfonic acid,acrylamido-2-methyl-1-propane-sulphonic acid), metal chelators (e.g.,styrene iminodiacetic acid), acrylamides and derivatives (e.g., N-methylacrylamide), methacrylamides and derivatives (e.g., N,N-dimethylacrylamide, N-(3-aminoprpoyl) methacrylamide), alkenes (e.g.,4-pentenoic acid, 3-chloro-1-phenyl-1-propene) (meth)acrylic acidanhydride and derivatives (e.g., methacrylic anhydride),silicon-containing monomers (e.g., (3-methacryloxypropyl) trimethoxysilane, tetramethyldisiloxane), polyenes (e.g., isoprene,3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene), azides (e.g.,4-azido-2,3,5,6-tetrafluorobenzoic acid), thiols (e.g., allylmercaptan). Acrylate terminated or otherwise unsaturated urethanes,carbonates and epoxies can also be used in embodiments of the presentinvention, as can silicon-based monomers.

If utilized, one or more crosslinking agents will preferably be one orseveral polymeric or oligomeric compounds, or a compound that providesfor cleavage under specific conditions. Crosslinking agents that lendrigidity to the subject polymeric compounds are known to those skilledin the art, and include, but are not limited to, di-, tri-, tetra- andpenta-functional acrylates, methacrylates, acrylamides, vinyls, allyls,and styrenes. Specific examples of cross-linking agents include but arenot limited to p-divinylbenzene, ethylene glycol dimethacrylate(abbreviated as EGDMA), tetramethylene dimethacrylate (abbreviated asTDMA), N,N′-methylene bisacrylamide(MDAA),N,N′-1,3-phenylenebis(2-methyl-2-propenamide)(PDBMP),2,6-bisacryloylamidopyridine, 1,4-diacryloyl piperazine (abbreviated asDAP), 1,4-phenylene diacrylamide, and N,O-bisacryloyl-L-phenylalaninol.Examples of reversible, cleavable crosslinkers include, but are notlimited to, N,N′-bis-(acryloyl) cystamine, N,N-diallyltartardiamide,N,N-(1,2-dihydroxyethylene) bisacrylamide, N1-((E)-1-(4-vinylphenyl)methylidene)-4-vinylanilene, allyl disulfide, andbis(2-methacryloyloxyethyl))disulfide. In preferred embodiments,ethylene glycol dimethacrylate is used as a cross-linking agent.Although the preferred cross-linking monomer is ethylene glycoldimethacrylate, embodiments of the present invention are not limited tothis agent, and other cross-linking monomers may be used, such as,divinylbenzene and trimethylolpropane trimethacrylate (abbreviated asTRIM).

Any ratio of simple monomers to crosslinking agents can be used thatprovides a MIP structure of appropriate integrity, e.g., that can beused in the context of the final application (e.g., in food or feedproducts, in water intended for aquaculture use, in vivo, etc). Thoseskilled in the art can select suitable ratios of monomers to provide thedesired structural integrity, which is intimately related to the natureand structure of the targeted molecule and to the nature and structureof the template used.

In embodiments, a MIP has a molar aflatoxin template to monomer ratio ofabout 100:1 to 1:100 (w/w). For example, ratios of aflatoxin template tomonomer ratios of about 1:2 to 1:7 are utilized. In other embodiments, aMIP has a molar monomer to crosslinker ratio of about 1:4 to 1:10.

In embodiments, a MIP changes volume when contacted with a solvent. Inembodiments, a MIP contacted with an aqueous solvent can adsorb up to 10times more water than its weight. In other embodiments, an MIP isselected that, when placed in a solvent, the volume of the MIP increasesabout 75%, 50%, 40%, 30%, 20%, 10%, 5% or less than the volume of theMIP in a dried state. The solvent or the solvent mixture used as amedium for MIP synthesis also has an impact on the swelling propertiesof MIP and on the size of cavities and pores size and distributionwithin the tri-dimensional MIP network and the formation of micro-,meso-, macrospheres and agglomerates. In embodiments, one or moreporogens may be employed in the synthesis of a MIP in order to alter thecavity size or swellability of the MIP. In certain embodiments, polarsolvents such as acetonitrile are used as a solvent or co-solvent forMIP polymerization when an increase in MIP swelling and increase of MIPcavity size is desired. Alternatively, such solvents are avoided when anincrease in MIP swelling and MIP cavity size is not desired (e.g., whenMIP is intended for use as a chromatographic column where swelling mayimpede flow rate and disturb the elution of analytes and the ability ofthe HPLC instrument to perform). In embodiments, the swellability of theMIP is compared to a corresponding NIP.

In embodiments, the characteristics of an MIP is compared to acorresponding NIP. A corresponding NIP comprises the same crosslinkedpolymer as the MIP but is formed in the absence of an aflatoxintemplate.

In embodiments, a composition comprises a MIP and a carrier. Inembodiments, the composition includes an effective amount of the MIP tosequester aflatoxin from a material. In embodiments, the effectiveamount is an amount that provides for the sequestering of at least 40%of the aflatoxin in the material based on weight per unit of material,and/or that reduces aflatoxin in the material to less than 0.5 parts perbillion (ppb). The compositions are formulated with suitable carriers,excipients, and other agents that provide suitable transfer, delivery,stability, and functionality of the MIP.

Methods of Synthesis of MIP

In embodiments, methods of synthesis of MIPs are described. Differentpolymerization methods may be used including free radical, cationic, andanionic polymerization. Polymerization conditions are selected andprovided herein that do not adversely affect the active conformation ofthe compound for which a complementary polymeric compound is to beproduced. In particularly preferred embodiments, free radicalprecipitation polymerization methods are used.

The method of making an MIP generally comprises providing an aflatoxintemplate, having a Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; R′ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,and a halo substituted C₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆alkoxy, and substituted C₁₋₆ alkyl; or wherein R₁ together with R₂ forma C₄₋₇ cycloalkyl ring, a halo substituted C₄₋₇ cycloalkyl ring, an oxosubstituted C₄₋₇ cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxysubstituted C₄₋₇ cycloalkyl ring and a carboxylic group substituted C₄₋₇cycloalkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl; and combining the aflatoxin template with at least one monomerand one or more crosslinkers. Upon combining the monomer andcrosslinker(s), the monomer and crosslinker(s) are polymerized to form amolecularly imprinted polymer intermediate.

A corresponding non-imprinted polymer (NIP) for a specific MIP is formedusing the same method, same monomer, and same crosslinker as the MIP butlacks the presence of the aflatoxin template.

The disclosure also provides compositions comprising a MIP as describedherein in a carrier. In embodiments, the carrier is a physiologicallyacceptable carrier. In other embodiments, the carrier is a solvent.

Molecularly Imprinted Polymer Intermediates

The aflatoxin template combined with a MIP precursor polymer forms amolecularly imprinted polymer intermediate. This molecularly imprintedpolymer intermediate is a complex of a crosslinked MIP precursorpolymer, having been made using a monomer, and an aflatoxin template. Inat least one embodiment, the aflatoxin template has or comprises aFormula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; R′ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl,and a halo substituted C₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆alkoxy, and substituted C₁₋₆ alkyl; or wherein R₁ together with R₂ forma C₄₋₇ cycloalkyl ring, a halo substituted C₄₋₇ cycloalkyl ring, an oxosubstituted C₄₋₇ cycloalkyl ring, C₄₋₇ cycloalkoxy ring a hydroxysubstituted C₄₋₇ cycloalkyl ring and a carboxylic group substituted C₄₋₇cycloalkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl. In particular, the aflatoxin template analog is selected from thegroup consisting of 4-(2-chloroethyl)-5,7-dimethoxy coumarin and5,7-dimethoxycyclo pentenon[2,3-c]coumarin.

In some embodiments, the aflatoxin template and at least one monomer andone or more crosslinkers is combined in one or more organic solvents. Inembodiments, one or more solvents are selected from the group consistingof acetonitrile, toluene, cyclohexane, polyvinyl alcohol in watersolution, and a mixture of two or more of acetonitrile, toluene,cyclohexane, polyvinyl alcohol in water solution. In a specificembodiment, a mixture of acetonitrile and toluene is used as a solvent.In particular, the solution of acetonitrile and toluene comprises atleast about 20% acetonitrile.

In other related embodiments, an initiator is used to generate freeradicals formed by thermal decomposition. Initiating agents include butare not limited to azo-bisisobutyronitrile (abbreviated as AIBN),azo-bisdimethylvaleronitrile (abbreviated as ABDV), dimethylacetal ofbenzil, benzoylperoxide (abbreviated as BPO), and4,4′-azo(4-cyanovaleric acid). In a specific embodiment,azo(bis)-isobutyronitrile is the initiator. In embodiments,polymerization is initiated by forming free radicals in an organicsolvent at a temperature between 55 and 110° C.

In embodiments, a method of synthesis further comprises adding a porogento change the size of the cavities and the swellability of the MIP.Examples of porogens include toluene, xylene, and ethylbenzene.

The molecularly imprinted polymer intermediate can be made using anaflatoxin template compound to monomer ratio from about 100:1 to 1:100.In other embodiments an aflatoxin template compound to monomer ratio isfrom about 1:2 to 1:7.

The molecularly imprinted polymer intermediate can also be made using acrosslinker, and the monomer to crosslinker ratio can be from about1:4.1 to 1:10.

Table 1 provides a series of examples of molecularly imprinted polymer(MIP) intermediates and non-imprinted polymer (NIP) intermediates andseveral aflatoxin template to monomer ratios and monomer to crosslinkerratios that are within the scope of the molecularly imprinted polymerintermediates described herein.

TABLE 1 Ratios of template vs. monomer vs crosslinker for thepreparation of 6 MIPs and NIPs. Mole Ratio Mole Ratio ProductTemplate:Mono- Monomer:Cross- Synthesis Mass Name mer linker Yield (g)MIP-001 1:2.0 1:5.7 50.2% 1.36 NIP-001 — 1:5.8 91.3% 4.93 MIP-002 1:2.31:9.6 76.3% 3.50 NIP-002 — 1:9.2 89.4% 4.14 MIP-003 1:4.6 1:4.1 62.1%3.03 NIP-003 — Not Synthesized N/A 0.00 MIP-004 1:4.0  1:10.0 74.7% 6.22NIP-004 — 1:10  93.4% 7.78 MIP-005 1:6.8 1:5.8 117.3%  13.54 NIP-005 —1:5.8 98.0% 11.31 MIP-006 1:7.1 1:9.6 69.3% 7.19 NIP-006 — 1:9.4 57.7%5.33

Once the molecularly imprinted polymer intermediate is formed, it isprecipitated and the aflatoxin template is removed from the molecularlyimprinted polymer intermediate to form a molecularly imprinted polymer.This process can be achieved by washing the molecularly imprintedpolymer intermediate with a solvent. In embodiments, a solvent isselected that has a similar polarity and/or solubility as the aflatoxintemplate. In embodiments, an organic solvent is selected from the groupof ethyl alcohol, methyl alcohol, acetonitrile, toluene, and a mixtureof thereof. Aflatoxin template removal can be determined by knownmethods such as by LC-MS. Upon removal of the aflatoxin template, amolecularly imprinted polymer is formed and available to sequester anaflatoxin molecule. In embodiments, the MIP is dried.

In embodiments, yield of the MIP can be enhanced by increasing thetemplate to monomer ratio and/or increasing the monomer to crosslinkerratio. In embodiments, the template to monomer ratio is at least 1:2and/or the monomer to crosslinker ratio of at least 1:6.

Method of Use

The disclosure provides methods of sequestering one or more aflatoxinscomprising contacting a molecularly imprinted polymer comprising acrosslinked polymer having a plurality of cavities, wherein at leastsome of the cavities provides for reversible binding to at least one ofthe aforementioned aflatoxins. Once the MIP is formed, it can be placedwithin or on a material suspected of containing an aflatoxin, optionallycontaining an aflatoxin, or known to contain an aflatoxin. It should beappreciated that the materials containing aflatoxin could be a gas,semi-gas, liquid, semi-liquid, or solid. In exemplary embodiments, thematerials containing aflatoxin are selected from the group consisting ofsoil, a spice, a beverage, a foodstuff, an animal feed, a pharmaceuticalcomposition, a nutraceutical composition, and a cosmetic composition. Inone embodiment, the material containing aflatoxin is milk.

A select amount (e.g. effective amount, or inclusion rate) of MIP isexposed to the material containing or suspected of containing aflatoxin.In embodiments, an amount of the MIP per unit of material is at least0.01%. For example, an MIP synthesized with a molar aflatoxin templatecompound to monomer ratio of at least 1:6.8, a molar monomer tocrosslinker ratio of at least 1:5.8, and has an inclusion rate of atleast 0.1%, adsorbs at least 76.5% of the aflatoxin M1(AFM1) from a 100ng/L AFM1 solution in buffer. In another example, an inclusion rate ofat least 1.0% showed 100% adsorption of AFM1. In other embodiments, theMIP/material ratio is at least 0.01% to 100%. In embodiments, an amountof MIP per volume of liquid is about 100 mg to 1 kilogram per liter ofmaterial.

In embodiments, a MIP is contacted with the material containing orsuspected of containing aflatoxin for at least 1 second. In otherembodiments, the MIP is contacted with the material containing aflatoxinor suspected of containing aflatoxin for about 1, 2, 3, 4, 5 minutes ormore. In other embodiments, the MIP is contacted with the material fromabout 1 second to 500 minutes.

In embodiments, the material and the MIP are contacted in a solutionwith a pH of 1-13. In other embodiments, the pH is about pH 6.0, pH 7.0,pH 7.5, or less.

In embodiments, the MIP is contacted with the material in batch with orwithout agitation. In other embodiments, an MIP is placed in achromatography column, such as solid phase extraction column.

Once the MIP is in contact with the material for a predetermined periodof time, the MIP, which now contains sequestered aflatoxin, is separatedfrom the material. One such separation method is filtration. Anotherseparation method is centrifugation.

Adsorption of the aflatoxin by the MIP ranges from at least 10%, 20%,30, or 40% or greater of the weight of an aflatoxin per unit ofmaterial. Adsorption obtained from a material is specific to theconditions used in terms of pH, temperature, concentration of toxin,nature of MIP, agitation, and flow of the material. If time of exposureof the MIP to the mycotoxin is increased and/or the inclusion rate isincreased, then a 100% adsorption is observed. Adsorption is affected bytime of exposure, concentration of aflatoxin, inclusion level of theMIP, and environment. When the material is exposed to the MIP for atleast 5 minutes, with an inclusion rate of at least 0.1%, the MIP cansequester at least 40% by weight of the aflatoxin in the material. Inrelated embodiments, the MIP will sequester a sufficient amount ofaflatoxin from the material to reduce the amount of aflatoxin in thematerial to less than 0.5 or less than 0.05 parts per billion.

In some embodiments, the material can be contacted with a MIP formultiple exposures until aflatoxin levels are reduced. For example, afirst exposure of the material to a MIP can remove about 10% or more ofthe aflatoxin. The MIP with bound aflatoxin is then removed and washedand reused or MIP with little or no bound aflatoxin is then contactedwith the material again. Multiple exposures can continue until theamount of aflatoxin is reduced for example, to less than 0.5 ppb.

Optionally, after separation of MIP with bound aflatoxin, aflatoxin canbe removed from the MIP by treating with a solvent that can disrupt thechemical association of the aflatoxin with the MIP. However, there is abalance between affinity of the MIP for binding of the aflatoxin and theamount of bound aflatoxin that can be removed. In embodiments, for a MIPwith high affinity for aflatoxin, the MIP releases about 25%, 20%, 15%,10%, 5%, 1% or less of the aflatoxin sequestered from the material inthe presence of a solvent, for example, as compared to a correspondingNIP. In certain embodiments, it is desirable to reuse a MIP from whichaflatoxin previously sequestered has been removed beforehand accordingto suitable and sufficient amount of organic solvent washes so that nodetectable amount of aflatoxin can be found leaching from the MIPmaterial using conventional LC-UV or LC-fluorescence, or LC-MSquantitative methodologies.

Another optional step in the process of using MIPs for the sequesteringof aflatoxin, is to detect the amount of aflatoxin (i.e. parts perbillion (ppb)) in a material prior to treatment with an MIP.Additionally, the material may be again tested, during and/or aftertreatment with an MIP to determine sequester rate of the aflatoxin.Furthermore, the amount of MIP required to sequester a pre-determinedconcentration of aflatoxin may also be elucidated, depending on theparticular MIP utilized. Moreover, the MIP complexed with aflatoxin,once separated from the material, may be tested for aflatoxinconcentration sequestered.

Quantitative adsorption efficacy can be determined by usingUPLC-Xevo-TQD MS/MS (Waters Corp.). For example, a gradient ofwater/0.1% formic acid (v/v) and methanol/0.1% methanol (v/v) is usedand analytes can be separated on an Acquity UPLC® BEH C18 1.7 μm 2.1×50mm column (Waters. Corp.). The method is optimized for the analysis ofAFM1/AFB1/aflatoxin template in buffer and milk using a C13-AFB1isotopic dilution and normalization technique.

EXAMPLES Synthesis of Aflatoxin M1 Template Molecules Example 1Preparation of 4-(2-chloroethyl)-5,7-dimethoxycoumarin (AFM-Template-1)

Cold solution of ethyl-4-chloroacetoacetate (26.6 gr) in acetic acid(12.5 ml), and concentrated sulfuric acid (6.25 ml) was added drop-wisefor 15 minutes to a solution of 3,5-dimethoxyphenol (25.0 gr) in aceticacid (50.0 ml) at 8-10° C. under nitrogen atmosphere. The reactionmixture was consecutively stirred at 20-25° C. for 1 hour, slowly heatedto 60° C. and stirred for 12 hours at 55-60° C. The reaction mixture wascooled to 40° C. and hot water (150.0 ml) was added drop-wise over aperiod of 30 minutes at 40-45° C. The mixture was cooled to roomtemperature and stirred for 1 hour to precipitate the product. Theproduct was filtered, washed with water (2×25 ml) and dried undersuction for 30 minutes. Cold methanol (50.0 ml) was added to the crudeproduct and the slurry was stirred at 8-10° C. for 30 minutes. Theproduct was filtered and washed with cold methanol (2×25 ml) and driedunder vacuum to obtain the final product,4-(2-chloroethyl)-5,7-dimethoxycoumarin (AFM-Template-1), which had theappearance of a white fluffy powder (39 gr). The resulting product wascarried forth and used in the next step as is.

Example 2 Preparation of4-(2,2-dicarboethoxy-ethyl)-5,7-dimethoxycoumarin (AFM-Intermediate-1)

Diethylmalonate (32.75 gr) was added to a mixture of4-(2-chloroethyl)-5,7-dimethoxycoumarin (AFM-Template-1, 40.0 gr),18-Crown-6 (4.96 gr), and potassium iodide (3.12 gr) in acetonitrile(400 ml) at room temperature under nitrogen atmosphere.Potassium-t-butoxide (t-BuOK, 22.8 gr) was added in one lot to thereaction mixture (slightly exothermic) at room temperature. Thetemperature of the reaction mixture (suspension) was slowly increased to40° C., and then stirred for 24 hours at 35-40° C. under nitrogenatmosphere. The reaction mixture was cooled to room temperature andevaporated to dryness under vacuum at 35-40° C. to produce a yellowsemi-solid residue. The residue was dissolved in a mixture of water (200ml) and ethylacetate (400 ml) under stirring. The pH of the mixture wasadjusted to 5 with diluted hydrochloric acid. The organic layer wasseparated from the aqueous layer, this latter being further extractedwith ethylacetate (2×200 ml). Organic layer dried over anhydrous sodiumsulfate (100 gr) were combined and filtered. The filtrate wasconcentrated to dryness under vacuum at 35-40° C. to give the4-(2,2-dicarboethoxy-ethyl)-5,7-dimethoxycoumarin (AFM-Intermediate-1),which had the appearance of a yellow solid (58 gr). The resultingproduct was carried forth and used in the next step as is.

Example 3 Preparation of Diacid (AFM-Intermediate-2)

Sodium hydroxide pellets (15.6 g) were added to a suspension of4-(2,2-dicarboethoxy-ethyl)-5,7-dimethoxycoumarin (AFM-Intermediate-1,58.0 g) in ethyl alcohol (290 ml) at room temperature. The temperatureof the reaction mixture (suspension) was slowly increased to 60° C., andthen stirred for 3 hours at 60-65° C. The reaction mixture was cooled toroom temperature and then pH of the mixture adjusted to 2 withconcentrated hydrochloric acid to precipitate the product. The slurrywas cooled to a temperature of 10° C. and stirred for 1 hour at 8-10° C.to complete precipitation of the product. The product was filtered(Crop-1), and then ethanol distilled-off from the mother liquor bydistilling at 20-25° C. under vacuum, and then the concentrated mass wascooled to 10° C. to precipitate the product, filtered the same (Crop-2).The combined product was washed with 1:1 (v/v) mixture of methanol andwater (2×200 ml) and then further dried under vacuum to obtain thediacid (AFM-Intermediate-2), which had the appearance of a yellow solid(45 g). The resulting product was carried forth and used in the nextstep as is.

Example 4 Preparation of Monoacid (AFM-Intermediate-3)

The diacid (AFM-Intermediate-2, 30 g) was suspended in m-xylene (300 ml)at room temperature. The temperature of the reaction mixture(suspension) was slowly increased to 135° C., and then stirred for 12hours at 135-140° C. The reaction mixture was cooled down to roomtemperature and then the formed precipitated filtered. The precipitatewas washed with n-Hexanes (2×100 ml) and dried under vacuum to obtainthe monoacid (AFM-Intermediate-3), which appeared as half-white solid(25 g). The resulting product was carried forth and used in the nextstep as is.

Example 5 Preparation of 5,7-dimethoxycyclo pentenon[2,3-c]coumarin(AFM-Template-2)

The monoacid (AFM-Intermediate-3, 4.75 g) was suspended inpolyphosphoric acid (9.50 gr) at room temperature under nitrogenatmosphere. The temperature of the reaction mixture (suspension) wasslowly increased to 75° C., and then stirred for 12 hours at 70-75° C.The reaction mixture was cooled to room temperature and then water (50ml) was added slowly to decompose the excess polyphosphoric acid and thereaction mixture was stirred for 1 hour at room temperature.Dichloromethane (50 ml) was added to the reaction mixture and stirredfor 15 minutes, organic layer was separated. The product was extractedwith dichloromethane (2×25 ml). The combined organic layer was driedover anhydrous sodium sulfate (25 g) and concentrated to dryness bydistillation under vacuum. The residue was suspended in methanol andstirred for 30 minutes at room temperature. The product was filtered andwashed with methanol (2×10 ml) and then dried under vacuum to obtain5,7-dimethoxycyclo pentenon[2,3-c]coumarin (AFM-Template-2), whichappeared as half-white solid (2.5 g).

Example 6 Produced MIP Composition and Characteristics

Experiments were conducted during development of embodiments of thedisclosure to test MIP polymers under their free flowing powder form fortheir adsorption properties toward AFM1 (Biopure, Romer Labs® Inc,Union, Mo.) mycotoxin and for the removal of the AFM1 mycotoxin fromliquid or semi-liquid media via chemical interactions. The MIP producedwas used herein to depict the differences in affinity of sequestrationof the AFM1 mycotoxin and to evaluate the specificity of the material.

Six independent MIPs were prepared using AFT-1 (1.0 mmol, template),methacrylic acid (2.0 mmol, MAA, monomer), and ethylene glycoldimethacrylate (5.0 mmol, EGDMA, cross-linker) in a mixture ofacetonitrile and toluene (1:3 v/v) at room temperature under nitrogenatmosphere by using different molar ratio of AFT-1 vs. MAA and monomervs. cross-linker (Table 1). The solution was stirred for 1 h at RT underinert atmosphere. Then, the azo(bis)-isobutyronitrile (0.01 mmol, AIBN,initiator) was added and slowly heated and maintained for 30 min at60-65° C. to precipitate the MIP microspheres. Two independentNon-Imprinted Polymers (NIP's) were also prepared through the sameprocedure but in the absence of AFT-1. The template (AFT-1) was removedfrom the MIP by continuous washing with toluene until completedisappearance of template in the washings as determined by the analysisof eluent through LC-UV, LC-fluorescence.

The resulting MIP and NIP polymeric material was synthesized as a blockpolymer which was ground to a powder with a mortar and pestle. The NIPpolymeric material was white in color and when ground to a powder washighly electrostatic. The MIP polymeric materials were brown in colordue to the presence of the brown colored template with the exception ofMIP-005 which was red in color (a different synthesis batch template wasused for this MIP which was red in color). Minimal color change wasexperienced during the toluene rinses of the MIP products. However whenwashed with methanol, the color of the powder was extremely muted andless dark as the colored template was rinsed from the polymer structure.The MIP polymeric materials in the powder form were also somewhatelectrostatic, although not to the degree of the NIP products.

Swelling properties of powder forms of MIP/NIP were investigated (Table2). We concluded that the swelling properties of MIP were considerablyhigher than NIPs. MIP-001 exhibited the greatest volume increase byswelling to 240% of its original size in buffer. MIP-002 also showedsignificant size increase to 200% of its original size. MIP-005 andNIP-005 were the only polymeric materials which showed no size increasewhen exposed to buffer for an extended period of time while NIP-004showed a minimal 11% volume increase. The remaining MIP products allexhibited a moderate degree of volume increase due to swelling, to150-167% of their original size.

TABLE 1 Ration of template vs. monomer vs cross linker for thepreparation of 6 MIPs and Nips. Mole Ratio Mole Ratio ProductTemplate:Mono- Monomer:Cross- Synthesis Mass Name mer linker Yield (g)MIP-001 1:2.0 1:5.7 50.2% 1.36 NIP-001 — 1:5.8 91.3% 4.93 MIP-002 1:2.31:9.6 76.3% 3.50 NIP-002 — 1:9.2 89.4% 4.14 MIP-003 1:4.6 1:4.1 62.1%3.03 NIP-003 — Not Synthesized N/A 0.00 MIP-004 1:4.0  1:10.0 74.7% 6.22NIP-004 — 1:10  93.4% 7.78 MIP-005 1:6.8 1:5.8 117.3%  13.54 NIP-005 —1:5.8 98.0% 11.31 MIP-006 1:7.1 1:9.6 69.3% 7.19 NIP-006 — 1:9.4 57.7%5.33

TABLE 2 Percent volume expansion of each MIP/NIP powder after 90 hexposure to pH 6.0 ammonium acetate buffer solution in NMR tubes.Product Percent Volume Increase MIP-001 140%  MIP-002 100%  MIP-003 67%MIP-004 50% MIP-005  0% MIP-006 67% NIP-004 11% NIP-005  0%

Example 7 Produced MIP Sequestration Capabilities TowardMycotoxins—Applied to AFM1 in Buffer

Quantitative adsorption efficacy was carried out using UPLC-Xevo-TQDMS/MS (a.k.a., UPLC-MS/MS) (Waters Corp.). A gradient of water/0.1%formic acid (v/v) and methanol/0.1% methanol (v/v) was used and analyteswere separated on an Acquity UPLC® BEH C18 1.7 μm 2.1×50 mm column(Waters. Corp.). The method was optimized for the analysis ofAFM1/AFB1/AFT-1 in buffer and milk using a C13-AFB1 isotopic dilutionand normalization technique.

Instant Trapping properties

Experiments were conducted during development of embodiments of thedisclosure to test for the inclusion rate of the MIP/NIP investigated byramping said levels of inclusion from 0.001 to 1.0% (w/v) of material ina pH 6.0 environment. Several instant trapping studies were done toascertain the viability of using MIP products to adsorb AFM1. To performthis study, 0.01 mg, 0.1 mg, 1.0 mg, and 10.0 mg of MIP-005 were loadedinto extraction cartridges with polytetrafluoroethylene (PTFE) fritsusing a slurry technique in buffer for the lowest inclusion rates.Briefly, MIP was put in suspension using buffer and loaded onto thecartridge and weighted to determine the precise amount of the MIP. Thequantities of MIP used in this experiment represent inclusion rates of0.001%, 0.01%, 0.1%, and 1.0% (w/v). This experiment was performed atroom temperature.

The polymeric material was “primed” by adding and subsequently eluting 1volume (1 mL) of water, 1 of methanol, and 2 of buffer in succession.One milliliter of a solution of buffer spiked with 100 ng/L of AFM1 wasthen added to each cartridge and followed after 1 min by 1 mL of bufferwith no AFM1. These final two elutions were collected in the samesilanized UPLC vial for analysis of AFM1 content. A volume of 1 mL ofmethanol was added to the cartridges for the elution of trapped AFM1 andthe eluent was collected followed by 1 mL of toluene eluent which waslikewise collected separately for analysis. Methanol and toluene eluentsamples were dried using nitrogen gas and reconstituted in 1 mL ofbuffer before analysis. To allow for effective quantification of resultsusing the UPLC-MS/MS, standards were created of known concentrations ofAFM1 in buffer at 1, 5, 10, 50, and 100 ng/L.

Results showed that 1 mg/L of free flowing polymer was sufficient forthe adsorption of 76.5% toward 100 ng/L of AFM1, which was selected aspotential aflatoxin target. FIG. 1. This inclusion rate was used as areference for the rest of the MIP evaluation. Instant sorption of 100ng/L of AFM1 by MIP and NIP packed into solid-phase extraction (SPE)cartridges and eluted with a 100% methanol solution was investigated. Wefound that the adsorption varied between 75.2 and 94.4 ng/L of AFM1adsorbed.

The quantity of AFM1 present in the methanol and toluene extractionrinses serves as an indicator of the strength with which the AFM1 isbeing held by the MIP/NIP. We are demonstrating that each product testedreleased between 31.1 and 44.4 ng/L of AFM1 when washed with 100%methanol with two exceptions. MIP-001 released 64.3 ng/L of AFM1 andMIP-005 released a low 22.6 ng/L of AFM1.

However, with the exception of these two products, each of the MIPs andNIPs exhibited a similar degree of interaction strength with the AFM1.Little to no AFM1 was found in the toluene rinses for each of theMIP/NIP products. This is likely due to the fact that much of the AFM1was released in the methanol extraction and also that the nonpolarnature of toluene had little effect on desorption of the polar AFM1. SeeFIG. 1.

Kinetic of Adsorption

Experiments were conducted during development of embodiments of thedisclosure to test the time of reaction and its effect on the adsorptionusing free flowing MIP/NIP reacted under 225 rpm orbital shaking at pH6.0 over 6 periods of time, from 5 to 500 minutes with a 90 ng/L AFM1 10mL solution. Adsorption efficacy was measured by quantitation of themycotoxin remaining in the supernatant and eluting from the MIP/NIPafter methanol wash, which defined adsorption efficacy and selectivity.Due to the fact that there is instant adsorption, the AFM1 adsorptionquantities for each time point (15, 30, 60, 90 minutes and 18 hrs) wereaveraged for each product. All test tubes were then centrifuged for 10minutes at 3,000 rpm and a transferred into UPLC vial for analysis. Thepowder (MIP or NIP) was then transferred to a 2 mL Eppendorf tube where1 mL of methanol was added and vortexed for approximately three seconds.Each tube was then centrifuged for 5 minutes at 10,000 rpm and 500 μL ofliquid was removed and placed in a UPLC vial. The samples were thendried using nitrogen and reconstituted in 500 μL of buffer beforeanalysis by means of a UPLC-MS/MS system.

All polymers at every time point were able to adsorb between 45 and 55%of AFM1 and non-significant differences were observed between MIP andNIP. See FIG. 2. Selectivity was however vastly different between MIPand NIP. MIPs depending on their formulation were only partiallyreleasing AFM1. The differences observed in terms of release of thetargeted molecule following methanol wash with the previous experiment,clearly demonstrated that the increase of the time of reaction betweentarget molecule and MIP increased the stability of the sequestering,whereas NIP showed a lower stability of interaction resulting in therelease of more of the targeted compound in the methanol wash. Thisexperiment established the clear specificity of the interaction andadsorption quality of the MIP toward aflatoxin M1. Further investigationdemonstrated that when different concentration of AFM1 were testedvarying from 45 to 450 ng/mL, MIP/NIP exhibited similar adsorptioncapacity around 50%, accounting for a chemical equilibrium betweenadsorbed vs. non-adsorbed AFM1 present in the environment.

Example 8 Produced MIP Sequestration Capabilities TowardMycotoxins—Applied to AFM1 in Milk

Experiments were conducted during development of embodiments of theinvention to test the MIP (MIP-003 for its capacity at interacting with225 ng/L AFM1 in raw milk. A slurry of MIP-003 in LC-MS grade water wasused to place 0.1 mg, 1.0 mg, and 10.0 mg of powder in respectivesilanized test tubes (1 mL of water from the slurry in each test tube).1 mL of water was placed in two additional test tubes with no powder toserve as controls in the form of both spiked and blank raw milk. 9 mL ofraw milk spiked to 250 ng/L AFM1 was then placed in each test tube,except for the blank milk control which received 9 mL of milk which wasnot spiked. All test tubes were then placed on an orbital shaker set to200 rpm for one hour at room temperature following which the test tubeswere centrifuged at 4,000 rpm for 10 minutes. Fourteen 100 mg C18 SPEcartridges (triplicate for each spiked milk sample and duplicate for theblank milk) were activated by eluting 1 mL of methanol and 1 mL of waterin succession using vacuum pressure. After centrifugation, 1 mL ofliquid was placed in each respective SPE cartridge and each sample, withthe exception of the blank milk, was spiked with 10 μL of a 100 ppbsample of AFB1 in acetonitrile to serve as an internal standard. Theliquids were then eluted using vacuum pressure followed by the elutionof 1 mL of water through each cartridge. Following this, 1 mL ofmethanol was placed in each cartridge and eluted into a silanized UPLCvial using positive pressure. After elution, one of the two blank milksamples was spiked to 225 ng/L AFM1 and with 10 μL of the 100 ppb AFB1internal standard solution. To allow for quantification of results,standards of 1, 5, 10, 25, 50, 100, 250, and 500 ng/L AFM1 concentrationwere created in acetonitrile. One milliliter of each sample was dried byblowing nitrogen over it and reconstituted in 1 mL of buffer. A volumeof 500 μL of each standard was then spiked with 5 μL of the 100 ppb AFB1solution to allow for the creation of a calibration curve.

At an inclusion rate of 0.1% (w/v), the MIP was able to remove 45.3% ofthe toxin. As seen in FIG. 3, we established that even 0.1 mg (0.001%inclusion rate) and 1.0 mg (0.01%) of free flowing powder also exhibitedadsorption in the range of 6.7% and 15.4% of AFM1 removed from milk,respectively. This experiment clearly defined the applicability of MIPat targeting specifically AFM1 in a complex raw milk matrix even atinclusion rates as small as 0.001%.

Example 9

An initial study evaluated the level of inclusion of MIP necessary tosequester AFM1. MIP-005 material was studied in buffer conditions. Asshown in the FIG. 5, it was found that 1 mg of powder (representing aninclusion rate of 0.1%, w/v) was able to adsorb 76.5% of the AFM1 fromthe 100 ng/L AFM1 solution in buffer. Meanwhile the 0.001% and 0.01%(w/v) inclusion rates resulted in negligible AFM1 adsorption. On theother hand, an inclusion rate of 1.0% (w/v) showed 100% adsorption ofAFM1. See FIG. 5.

All publications and patents described herein are hereby incorporated byreference.

Those skilled in the art will readily appreciate that many modificationsare possible without materially departing from the novel teachings andadvantages of this disclosure. Accordingly, all such modifications areintended to be included within the scope of this disclosure as definedin the following claims.

1.-42. (canceled)
 43. A molecularly imprinted polymer comprising acrosslinked polymer made from monomers which are chemically bonded toeach other, wherein the crosslinked polymer defines a plurality ofcavities, wherein at least one of the cavities was made using anaflatoxin template having Formula (I):

wherein R₁ is selected from H, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, and ahalo substituted C₁₋₆ alkyl; R₂ is selected from halo, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, a halo substituted C₁₋₆ alkyl, CH₂C(O)OR′, andCH(C(O)OR′)₂; wherein R′ is selected from H, C₁₋₆ alkyl, and substitutedC₁₋₆ alkyl; and R₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆alkyl, or wherein R₁ together with R₂ form a C₄₋₇ cycloalkyl ring, ahalo substituted C₄₋₇ cycloalkyl ring, an oxo substituted C₄₋₇cycloalkyl ring, C₄₋₇ cycloalkoxy ring, a hydroxy substituted C₄₋₇cycloalkyl ring and a carboxylic group substituted C₄₋₇ cycloalkyl; andR₃ is selected from H, C₁₋₆ alkoxy, and substituted C₁₋₆ alkyl, whereinthe at least one cavity is sized and/or shaped to allow an aflatoxin tobe boundable therein.
 44. The molecularly imprinted polymer of claim 43,wherein aflatoxin template is selected from the group consisting of4-(2-chloroethyl)-5,7-dimethoxy coumarin, 5,7-dimethoxycyclopentenon[2,3-c]coumarin, or any combination thereof.
 45. The molecularlyimprinted polymer of claim 43, wherein the monomers are methacrylicacid, 2-vinylpyridine, 2-hydroxyethylmethacrylate, or any combinationthereof.
 46. The molecularly imprinted polymer of claim 43, wherein thecrosslinked polymer comprises a polymer network formed by monomers and acrosslinker.
 47. The molecularly imprinted polymer of claim 43, whereinthe aflatoxin template and the monomers are used in a molar ratio offrom about 100:1 to about 1:100.
 48. The molecularly imprinted polymerof claim 46, wherein the monomers and the crosslinker are used in amolar ratio of from about 1:4.1 to about 1:10.
 49. The molecularlyimprinted polymer of claim 43, wherein the at least one cavity has acapacity to form a complex with an aflatoxin to bind the aflatoxintherein.
 50. The molecularly imprinted polymer of claim 43, wherein theaflatoxin template is an isolated compound selected from the groupconsisting of:

and combinations thereof.
 51. The molecularly imprinted polymer of claim43, wherein the aflatoxin template is an isolated compound that has aformula of: